Responsive elastic polymers and methods of making and using same

ABSTRACT

Disclosed herein are functionalized hyaluronic acid (HA), a responsive elastic polymer system comprising functionalized HA, and methods of fabrication and utilization of the same. This polymer system may be used for controlled local or systemic drug delivery release of analgesics, anesthetics, antibiotics and other drugs as well as tissue engineering articles

PRIORITY

This application is related to, claims the priority benefit of, and is acontinuation of U.S. application Ser. No. 16/309,141 filed Dec. 12,2018, which issues as U.S. Pat. No. 11,491,233 on Nov. 8, 2022, and isrelated to, claims the priority benefit of, and is a US National Phasefiling of International Patent Application No. PCT/US2017/037248, filedon Jun. 13, 2017, which claims the priority benefit of: (1) U.S.Provisional Application No. 62/349,475, filed on Jun. 13, 2016; and (2)U.S. Provisional Application No. 62/366,160, filed on Jul. 25, 2016. Theentire contents of each of the aforementioned priority applications areexpressly incorporated by reference herein in their entireties.

TECHNICAL FIELD

The present disclosure generally relates to polymer systems, and inparticular to functionalized hyaluronic acid (HA), a responsive elasticpolymer system which includes functionalized HA, and methods of itsfabrication and utilization.

BACKGROUND

This section introduces aspects that may help facilitate a betterunderstanding of the disclosure. Accordingly, these statements are to beread in this light and are not to be understood as admissions about whatis or is not prior art.

Controlled drug delivery offers numerous advantages compared toconventional dosage forms including improved efficacy, reduced toxicity,reduced need for specialized drug administration (e.g. repeatedinjections), and improved patient compliance and convenience [1].Several biomaterial-based controlled systems such as polymersomes,polymeric micelles, microspheres, nanospheres, nanoparticles, polymericfilms, and silica nanoparticles are currently investigated to deliverdrugs in a spatiotemporally controlled manner. Hydrogels composed ofpolymer networks swollen in water provide a promising delivery platformfor controlled drug release applications because depot formulations canbe created to allow drugs to slowly elute, maintaining a high localconcentration of drug in the surrounding tissues over an extended period[2]. In addition, such hydrogels may also be used for controlledsystemic drug release. Because responsive hydrogels that can respondsimultaneously to triggers, such as pH, temperature, light, ions, andprotein, they represent attractive candidate materials for developmentof drug delivery vehicles to achieve prolonged action [3].

However, the utility of conventional hydrogels for clinical applicationsis often hampered by their inferior mechanical performance. Forinstance, in general they are very weak and do not exhibit high stretchability [4]. Consequently, it presents a grand challenge to use theconventional hydrogels in load-bearing anatomic sites like synovialjoints. To function effectively in those settings, the hydrogel has tobe stretchable and expandable under compression and tension withoutbreaking. There is therefore an unmet need for the development ofresponsive, highly elastic injectable hydrogels.

SUMMARY

This disclosure provides a composition comprising a polymer matrixcomprising functionalized hyaluronic acid (HA) of at least 100 monomericunits cross-linked to at least one unit of a telechelic polymer.

In some embodiment, the aforementioned functionalized hyaluronic acid isselected from the group consisting of the formula of I-IV:

wherein R₁, R₂, R₃, R₄, and R₅ may include any one of or a combinationof haloacetates, dihydrazides, amines, thiols, carboxylic acids,aldehydes, ketones, active hydrogen sites on aromatic ring, dienes,azide isothiocyanates, isocyanates, acyl azides, N-hydroxysuccinimide(NHS) esters, sulfo-NHS, sulfonyl chloride, epoxides, carbonates, arylhalides, imidoesters, carbodiimides (e.g. N, N′-dicyclohexylcarbodiimide(DCC) and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC)),alkylphosphate compounds, anhydrides, fluorophenyl esters, hydroxymethylphosphines, guanidino groups, iodoacetyl derivatives, maleimides,aziridines, acryloyl derivatives, arylating agents, disulfidederivatives, vinylsulfone, phenylthioester, cisplatins, diazoacetates,carbonyl diimidazoles, oxiranes, N,N′-disuccinimidyl carbonates,N-hydroxylsuccinimidyl chloroformates, alkyl halogens, hydrazines,alkynes, and phosphorus-bound chlorine.

In some embodiment the aforementioned telechelic polymer is selectedfrom the group consisting of any one of or a combination of poly(aliphatic ester) (e.g. poly(lactide) (PLA), poly(ε-caprolactone) (PCL),poly(glycolic acid) (PGA), poly(lactic-co-glycolic acid) (PLGA),poly(trimethylene carbonate) (PTMC), polydioxanone (PDS), poly(orthoester), polyanhydrides, poly(anhydride-co-imide),poly(anhydride-esters), polyurethanes(e.g. degrapols), poly(amide),poly(esteramide), poly(orthoesters), poly(dioxanones), poly(acetals),poly(ketals), poly(carbonate), poly(orthocarbonates),poly(hydroxylbutyrates), poly(hydroxyl-valerats), poly(alkyleneoxalates), poly(alkylene succunates), poly(malic acid), poly(aminoacids), poly(vinylpyrolidone), poly(hydroxycellulose), poly(glycerolsebacate), poly(ethylene imine), poly(acrylic acid)(PAA),poly(N,N′-diethylaminoethyl methacrylate, polyethylene glycol (PEG),poly(propylene oxide) (PPO), PEO-b-PPO block copolymers (e.g. pluronics(or poloxamers), and tetronic), poly(vinyl alcohol) (PVA), poly(N-isopropylacrylamide) (PNIPAm), Poly(N,N-diethylacrylamide) (PDEAAm),poly(oxazolines) (e.g. poly(2-methyloxazoline andpoly(-ethyl-2-oxazoline), oligo(ethylene glycol) fumarates (OPFs),poly(propylene fumarate), poly(alkyl cyanoacrylates), poly(acrylicamide), synthetic poly(amino acids) (e.g. poly (L-glutamic acid) (L-PGA)and poly (aspartic acid)), polyphosphazenes, and poly(phosphoesters).

In some embodiment the aforementioned telechelic polymer is selectedfrom the group consisting of any one of or a combination of fibrin,collagen, matrigel, elastin, elastin-like peptides, albumin, naturalpoly (amino acids) (e.g. cyanophycin, poly (ε-l-lysine), poly(τ-glutamic acid)), polysaccharides (e.g. chitosan, dextran, chondroitinsulfate, agarose, alginate, methylcellulose, and heparin).

In some embodiment the aforementioned functionalized hyaluronic acidcomprises at least one monomeric unit of hyaluronic acid functionalizedwith an amine moiety.

In some embodiment the aforementioned functionalized hyaluronic acidcomprises at least one monomeric unit of hyaluronic acid functionalizedwith an acrylate moiety.

In some embodiment the aforementioned telechelic polymer is thiolatedpoly (N-isopropylacrylamide) PNIPAm.

This disclosure further provides a method of making a hyaluronic acid(HA) based polymer matrix comprising functionalized hyaluronic acid ofat least 100 monomeric units cross-linked to at least one unit of atelechelic polymer. The method comprising the steps of:

-   preparing a functionalized hyaluronic acid of at least 100 monomeric    units; preparing a prefabricated functional telechelic polymer;-   crosslinking the functionalized hyaluronic acid with the functional    telechelic polymer by carbodiimide-mediated reactions,    esterification, amidation, aldehyde and ketone reactions, active    hydrogen reactions, photo-chemical reaction, azide-alkyne    cycloaddition(e.g. copper-catalyzed azide-alkyne cycloaddition    (CuAAC), copper-free azide-alkyne huisgen cycloaddition),    thiol-click reaction, diels-alder reaction, nitrile oxide    cycloaddition, and an enzymatic crosslinking strategy (e.g.,    horseradish peroxidase and hydrogen peroxide).

In some embodiment the aforementioned functional telechelic polymer isthiolated PNIPAm.

In some embodiment the aforementioned HA based polymer matrix furthercomprises at least one monomeric unit of a polymer which can be preparedby a polymerization reaction of i) the ‘grafting to’ and (ii) the‘grafting from’ strategies.

In some embodiment the aforementioned crosslinking reaction between thefunctionalized hyaluronic acid and thiolated PNIPAm is a thiol-enereaction.

In some embodiment the aforementioned ‘grafting from’ method involvesthe functionalized hyaluronic acid composing at least one ofpolymerizable moiety, an initiator, a RAFT agent and an infertier.

In some embodiment the aforementioned polymerizable moiety is anacrylate.

In some embodiment the aforementioned RAFT agent isS-1-dodecyl-S′-(α,α′-dimethyl-α″-acetic acid) trithiocarbonate (DATC).

In some embodiment the aforementioned polymerization reaction is a‘grafting from’ method using surface initiated RAFT polymerization ofPNIPAm.

This disclosure further provides a polymer-based drug delivery platformcomprising aforementioned composition further encapsulating anesthetics,analgesics or antibiotic with the polymer matrix system through physicalinteractions or chemical interaction.

In some embodiment the aforementioned physical interactions comprise anyone of or a combination of hydrophobic interaction, hydrophilicinteraction, hydrogen bonding, and inter-molecular electrostaticinteractions.

In some embodiment the aforementioned drug delivery platform comprisesany one of opioids (e.g. morphine) or nonsteroidal anti-inflammatorydrugs (NSAIDs) including chloroprocaine, bupivacaine, lidocaine, andprocaine.

This disclosure further provides a method of controlled delivering adrug. The method comprising:

-   Preparing a polymer matrix comprising functionalized hyaluronic acid    (HA) of at least 100 monomeric units cross-linked to a telechelic    polymer with a theoretical substitution degree of a telechelic    polymer ranging from 100% to 30%;-   Preparing a polymer matrix comprising functionalized hyaluronic acid    (HA) of at least 100 monomeric units cross-linked to a telechelic    polymer with a theoretical substitution degree of amine group    ranging from 20% to 80%;-   Preparing the conjugation of the drug in the polymer matrix at a    concentration ranging from 0-30% (w/v); and-   Observing the drug release rate decreases with increasing molecular    weight of the polymer matrix.

In some embodiment, the aforementioned drug delivery polymer matrix isHA-g-PNIPAm and the drug is morphine.

These and other features, aspects and advantages of the presentinvention will become better understood with reference to the followingfigures, associated descriptions and claims.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a schematic illustration depicting synthetic pathways ofhyaluronic acid based polymer matrix.

FIG. 2 shows a functionalized monomeric hyaluronic acid represented byformula I, II, III, IV.

FIG. 3 shows the synthesis of HA-g-PNIPAm method via the combination ofreversible addition-fragmentation chain transfer (RAFT) polymerizationand thiol-ene click reaction.

FIG. 4 shows the synthesis of HA-g-PNIPAm via a ‘grafting from’ methodusing the reversible addition-fragmentation chain transfer (RAFT)polymerization.

FIG. 5 shows a schematic illustration of the advantages of HA-g-PNIPAmhydrogels.

FIG. 6 shows MIR spectra of HA (black line), HA-ADH (blue line), HA-NAS(pink line) and HA-g-PNIPAm(red line), demonstrating successfulsynthesis of each step.

FIG. 7 shows ¹H NMR spectra of HA, HA-ADH, HA-NAS and HA-g-PNIPAm,demonstrating successful synthesis of each step.

FIG. 8 shows reversibility of HA-g-PNIPAm hydrogels.

FIG. 9 shows HA-g-PNIPAm hydrogel forming confirmation.

FIG. 10 shows time course pictures of HA-g-PNIPAm hydrogel with loadingand unloading at 37° C., demonstrating the elastic nature of hydrogels.

FIG. 11 shows frequency sweeps of HA-g-PNIPAm hydrogels. The linearmodulus plateau with respect to frequency was determined. As theconcentration of HA-g-PNIPAm increase, the gel stiffness increases.

FIG. 12 shows a proposed mechanism of HA-g-PNIPAm gels formation: theformation of intra- and intermolecular hydrogen bonds.

FIG. 13 shows an in vitro release profile of morphine from morphineloaded HA-g-PNIPAm (13k) with the concentration (w/v) 5%, 7.5%, 10% and15% respectively, and HA-g-PNIPAm (13k) hydrogels control. Morphineconcentration is 0.468% (w/v). As the concentration of HA-g-PNIPAmincreases, slower release of morphine.

FIG. 14 shows Pharmacokinetics study of morphine in rats. (A) A carotidartery catheterization surgery; (B) Subcutaneous (s.c.) injection (0.25mL) of morphine-loaded 10% HA-g-PNIPAm hydrogels; (C) Blood samples werecollected points using an automated system (Culex) through preplacedcarotid catheter; and (D) Recovered tissues showing pockets of hydrogelswere visible after 3 days of subcutaneous injections (0.25 mL)confirming the hydrogel formation following subcutaneous injections.

FIG. 15 shows in vivo drug release profile following subcutaneousinjections (0.25 mL) in rats. The morphine plasma concentration (ng/mL)as a function of time. The blue line indicates the therapeutic plasmaconcentration (10 ng/mL).

FIG. 16 shows schematics of injectable hydrogels for intraarticulardelivery of an analgesic or anesthetic.

FIG. 17 shows frequency sweeps of HA-g-PNIPAm hydrogels at theconcentrations of 10% (w/v). The linear modulus plateau with respect tofrequency was determined. As the molecular weight of HA-g-PNIPAmincrease, the gel stiffness increases.

FIG. 18 shows (A) FTIR spectra of 10% PNIPAm (w/v) at a temperature of0° C., 31° C., and 37° C., respectively; (B) FTIR spectra of 10%HA-g-PNIPAm13 k (w/v) at a temperature of 0° C., 31° C., and 37° C.,respectively. The results indicate hydrogen bonding plays an importantrole in HA-g-PNIPAM hydrogel formation.

FIG. 19 shows an in vitro release profile of morphine from morphineloaded HA-g-PNIPAm 13k and 20k, respectively. Morphine concentration is0.468% (w/v). As the molecular weight of HA-g-PNIPAm increased, therelease of morphine decreased.

FIG. 20 shows data related to intra-articular injections into cadaverdogs. The hydrogels distributed well through the cranial compartment ofthe joint. The gel most likely will form thin sheets in the trochleargroove and on the lateral and medial side of the condyles.

FIG. 21 shows the influence of the grafting density and concentrationsof HA-g-PNIPAm on (A) gelation temperature and (B) gelation time.Theoretical substitution degrees (DS) of: S5 of 100%, S4 of 80%, S3 of64%, S2 of 40%, and S1 of 30% at the concentrations of 10% (w/v). S6: atDS of 100% and at the concentrations of 15%.

FIG. 22 shows tunable amine groups in HA-g-PNIPAm17k (Structure No. 5,No. 2, No. 6 and No. 4) with PNIPAm DS of 100%, 40%, 50%, and 80%respectively.

FIG. 23 shows injectability and degradation of HA-g-PNIPAm hydrogels (atthe concentration of 15% (w/v)) using a rat model. Photograph imagesshow (A) Intra-articular injections into a rat; (B) Hydrogel formedright after injections; (C) Hydrogel degraded 21-day post injection.

FIG. 24 shows intravital fluorescence imaging of the release of AlexaFluor 680 conjugated BSA from HA-g-PNIPAm hydrogels. (n=4).

FIG. 25 shows total radiant efficiency emanating from the knee joint asa function of time. Alexa Fluor 680 conjugated BSA (BSA-AF) releasedfrom the HA-g-PNIPAm hydrogels showed a maximum concentration post 6-12hours intra-articular injections and sustained release profile, and theBSA-AF was detected in the knee joints for more than 24 hours.

FIG. 26 shows ex vivo fluorescence imaging of different tissues at 72hours post injection.

DESCRIPTION

For the purposes of promoting an understanding of the principles of thepresent disclosure, reference will now be made to the embodimentsillustrated in the drawings, and specific language will be used todescribe the same. It will nevertheless be understood that no limitationof the scope of this disclosure is thereby intended.

While the concepts of the present disclosure are illustrated anddescribed in detail in the figures and the description herein, resultsin the figures and their description are to be considered as exemplaryand not restrictive in character; it being understood that only theillustrative embodiments are shown and described and that all changesand modifications that come within the spirit of the disclosure aredesired to be protected.

Unless defined otherwise, the scientific and technology nomenclatureshave the same meaning as commonly understood by a person in the ordinaryskill in the art pertaining to this disclosure.

As used herein, “the degree of substitution” (DS) of substituent groups,(e.g., PNIPAm or amine groups) is the (average) number of substituentgroups ((e.g., PNIPAm or a amine group) attached to total monomeric unitof polymers HA, for example, a theoretical DS of HA equals (number ofsubstituent groups)/(number of monomeric unit of polymers HA). Since permonomeric unit of polymers HA contains one —COOH, the DS=(number ofsubstituent groups)/(number of−COOH).

In response to the unmet need, the development of responsive, highlyelastic injectable hydrogels that include functionalized hyaluronic acid(HA) is disclosed herein.

HA is an immunoneutral polysaccharide consisting of alternatingdisaccharide units of [β(1,4)-D-glucuronicacid-β(1,3)-N-acetyl-D-glucosamine) linkages [5]. HA is the onlynon-sulfated glycosaminoglycan that is widely distributed throughout thebody, especially in the synovia of j oints, the corpus vitreum of theeyes, and the dermis of the skin. HA is predominantly localized to theextracellular and pericellular matrix. Functionally, HA contributes tothe elastoviscosity of fluid connective tissues including synovial fluidand vitreous humor, and modulates hydration and transport of waterthrough tissues [6]. The enzymatic degradation of HA results from theaction of three types of enzymes: hyaluronidase, β-glucuronidase, andβ-nacetyl-hexosaminidase, which mainly accounts for HA derivedhydrogel's biodegradable properties [7]. Additionally, HA has been usedclinically for more than thirty years [8, 9]. For example, it has beenapproved by the US Food and Drug Administration for the treatment ofosteoarthritis in humans since 1997 [10].

Cross-linked HA-based polymeric biomaterials are widely used for drugdelivery vehicles owing to the major advantages of HA including: (i)biodegradability and biocompatibility; (ii) ease of chemicalmodification due to an abundance of carboxylic acid and hydroxyl groups;(iii) high potential drug loading; (iv) its intrinsic targetingproperties, due to the selective interactions with receptors, such asCD44, Toll2, Toll4, RAAMM receptor or hyaluronan receptors forendocytosis. (vi) HA degraded from HA-based polymeric biomaterials mayserve as a lubricant and shock absorber in the joints. HA couldstabilize the joint function due to its lubrication properties at lowshear and increased friction at high shear. A thin layer of HA acts ashock absorber between cartilage and cartilage/meniscal surface. And(vii) HA coats pain receptors to prevent binding to peptide agonists[11, 12].

Cross-linked HA-based polymeric biomaterials may be used for tissueengineering due in part to their ability to efficiently encapsulatecells. Mechanical and structural properties may be manipulated bymodification of the crosslinking density which controls network poresize, water content, mechanical properties, and cell-materialinteractions. In some cases, cross-linked polymers or gels may have ahigh, tissue-like water content which may allow nutrient and wastetransport. HA has numerous useful biological properties within tissueintegration including wound healing [13], cell adhesion andproliferation [14], cell motility, angiogenesis, cellular signaling, andmatrix organization [9].

According to at least one embodiment, an HA-based polymer matrix can beprepared following two main strategies: (i) the ‘grafting to’ and (ii)the ‘grafting from’ strategies as shown in FIG. 1 .

The ‘grafting to’ strategy involves the attachment of prefabricatedpolymers via either physisorption or covalent bond formation(chemisorption) [15]. HA can be modified in many ways to alter theproperties of the resulting materials, including modifications leadingto hydrophobicity and biological activities [10]. In part, the presentdisclosure provides for a composition comprising at least one monomericunit of HA functionalized by at least one functional group moiety.Chemical modifications of HA can be targeted to three functional groups:the glucuronic acid carboxylic acid, the primary and secondary hydroxylgroups, and the N-acetyl group (following deamidation). In someembodiments, compositions of HA in the present invention are providedthat may be represent by Formula I, II, III and IV (FIG. 2 ).

Carboxylates in a HA backbond can be modified by carbodiimide-mediatedreactions, esterification, and amidation. Hydroxyls in a HA backbond canbe modified by etherify cation, divinylsulfone crosslinking,esterification, and bisepoxide crosslinking. Additionally, convertingdiols to aldehyde can be achieved through periodate oxidation of HA.Finally, deacetylation of the N-acetyl group of HA recovers an aminogroup which can then react with an acid using the same amidation.

The functional groups R₁, R₂, R₃, R₄ and R₅ comprise any one of or acombination of haloacetate, dihydrazide, amines, thiol, carboxylic acid,aldehyde, ketone, active hydrogen sites on aromatic ring, diene, azideisothiocyanates, isocyanates, acyl azides. N-hydroxysuccinimide (NHS)ester, sulfo-NHS, sulfonyl chloride, epoxides, carbonates, aryl halide,imidoesters, carbodiimides (e.g. N,N′-dicyclohexylcarbodiimide (DCC) and1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC)), alkylphosphatecompound, anhydride, fluorophenyl ester, hydroxymethyl phosphine,guanidino group, iodoacetyl derivative, maleimides, aziridines, acryloylderivatives, arylating agents, disulfide derivative, vinylsulfone,phenylthioester, cisplatin, diazoacetate, carbonyl diimidazole,oxiranes, N,N′-disuccinimidyl carbonate, N-hydroxylsuccinimidylchloroformate, alkyl halogens, hydrazine, maleimide, alkyne, andphosphorus-bound chlorine.

The telechelic polymers can be both synthetic polymers and naturalpolymers. Synthetic polymers comprise any one of or a combination ofpoly (aliphatic ester) (e.g. poly(lactide) (PLA), poly(ε-caprolactone)(PCL), poly(glycolic acid) (PGA), poly(lactic-co-glycolic acid) (PLGA),poly(trimethylene carbonate) (PTMC), polydioxanone (PDS), poly(orthoester), polyanhydrides, poly(anhydride-co-imide),poly(anhydride-esters), polyurethanes (e.g. degrapols), poly(amide),poly(esteramide), poly(orthoesters), poly(dioxanones), poly(acetals),poly(ketals), poly(carbonate), poly(orthocarbonates),poly(hydroxylbutyrates), poly(hydroxyl-valerats), poly(alkyleneoxalates), poly(alkylene succunates), poly(malic acid), poly(aminoacids), poly(vinylpyrolidone), poly(hydroxycellulose), poly(glycerolsebacate), poly(ethylene imine), poly(acrylic acid)(PAA),poly(N,N′-diethylaminoethyl methacrylate, polyethylene glycol (PEG),poly(propylene oxide) (PPO), PEO-PPO block copolymers (e.g. pluronics(or poloxamers), and tetronic), poly(vinyl alcohol) (PVA), poly(N-isopropylacrylamide) (PNIPAm), Poly(N,N-diethylacrylamide) (PDEAAm),poly(oxazolines) (e.g. poly(2-methyloxazoline andpoly(-ethyl-2-oxazoline), oligo(ethylene glycol) fumarates (OPFs),poly(propylene fumarate), poly(alkyl cyanoacrylates), poly(acrylicamide), synthetic poly(amino acids) (e.g. poly (L-glutamic acid) (L-PGA)and poly (aspartic acid)), polyphosphazenes, poly(phosphoesters) andblends thereof.

The synthetic telechelic polymers can be prepared by conventionalmethods such as mass polymerization, solution (or homogeneous)polymerization, suspension polymerization, emulsion polymerization,radiation polymerization (using y-ray, electron beam or the like). Thesynthetic telechelic polymers can be prepared by addition or chaingrowth polymerization, coordination polymerization, condensation or stepgrowth polymerization. Addition or chain growth polymerization includesfree radical polymerization, controlled-living radical polymerization(e.g. atom transfer radical polymerization (ATRP), reversible additionfragmentation transfer (RAFT) polymerization, and nitroxide-mediatedradical polymerization (NMP)), cationic polymerizations, anionicpolymerizations and the like.

The natural polymers comprise any one of or a combination of fibrin,collagen, matrigel, elastin, elastin-like peptides, albumin, naturalpoly (amino acids) (e.g. cyanophycin, poly (ε-l-lysine), poly(τ-glutamic acid)), polysaccharides (e.g. chitosan, dextran, chondroitinsulfate, agarose, alginate, methylcellulose and heparin), and blendsthereof.

The conjugation of functional HA and telechelic polymers can be achievedby carbodiimide-mediated reactions, esterification, amidation, aldehydeand ketone reactions, active hydrogen reactions, photo-chemicalreaction, azide-alkyne cycloaddition (e.g. copper-catalyzed azide-alkynecycloaddition (CuAAC), copper-free azide-alkyne huisgen cycloaddition),thiol-click reaction, diels-alder reaction, nitrile oxide cycloaddition,and an enzymatic crosslinking strategy (e.g., horseradish peroxidase andhydrogen peroxide).

The conjugated linkages comprise any one of or a combination ofisothiourea, isourea, amide, sulfonamide, shift-base, secondary amine,carbamate, arylamine, amidine, phosphoramidate, thioether, disulfide,β-thiosulfonyl, ester, carbamate, hydrazone, diazo, triazoles,carbohydrates, and amino acid esters bond.

Alternatively, a cross-linker HA-based polymer matrix can be producedvia a ‘grafting from’ method (FIG. 1 ). A ‘grafting from’ methodinvolves preparing the precursor to the backbone polymer with monomerunits that contain functionalities ultimately capable of initiatingpolymerization of a second monomer [15]. In the embodiment, at least oneof the monomeric units of HA is conjugated to at least one ofpolymerizable moiety, an initiator, an RAFT agent and an iniferter.

A polymerizable moiety includes any moiety that is capable ofpolymerizing upon exposure to a polymerizing initiator. A polymerizablemoiety may include alkenyl moieties such as acrylates, methacrylates,dimethacrylates, oligoacrylates, oligomethoacrylates, ethacrylates,itaconates acrylamide, aldehydes, ethylenically unsaturated monomers.Ethylenically unsaturated monomers may include alkyl esters of acrylicor methacrylic acid, the nitrile and amides of the same acids,unsaturated monomers containing carboxylic acid groups, andpolyethylenically unsaturated monomers. Examples of alkyl esters ofacrylic or methacrylic acid are methyl methacrylate, ethyl methacrylate,butyl methacrylate, ethyl acrylate, butyl acrylate, hexyl acrylate,n-octyl acrylate, lauryl methacrylate, 2-ethylhexyl methacrylate, nonylacrylate, benzyl methacrylate, the hydroxyalkyl esters of the same acidssuch as 2-hydroxyethyl acrylate, 2-hydroxyethyl methacrylate, and2-hydroxypropyl methacrylate. Examples of the nitrile and amides of thesame acids are acrylonitrile, methacrylonitrile, and methacrylamide,vinyl acetate, vinyl propionate, vinylidene chloride, vinyl chloride,and vinyl aromatic compounds such as styrene, t-butyl styrene and vinyltoluene, dialkyl maleates, dialkyl itaconates, dialkylmethylene-malonates, isoprene, and butadiene. Examples of unsaturatedmonomers containing carboxylic acid groups include acrylic acid,methacrylic acid, ethacrylic acid, itaconic acid, maleic acid, fumaricacid, monoalkyl itaconate. Example of polyethylenically unsaturatedmonomers include butadiene, isoprene, allylmethacrylate, diacrylates ofalkyl diols (e.g. butanediol diacrylate and hexanediol diacrylate, anddivinyl benzene).

A RAFT agent moiety includes any moiety that is capable of trapping apropagating polymer radical and releasing a polymer fragment as aradical to achieve highly controlled polymerizations [16]. A RAFT agentmoiety may include dithiobenzoates (e.g. cumyl dithiobenzoate,cyanopentanoic acid dithiobenzoate), trithiocarbonates (e.g.4-cyano-4-(dodecylsulfanylthiocarbonyl) sulfanyl pentanoic acid,phthalimidylmethyl trithiocarbonates,S-1-dodecyl-S′-(α,α′-dimethyl-α″-acetic acid) trithiocarbonate (DATC),3,5-bis(2-dodecylthiocarbonothioylthio-1-oxopropoxy) benzoic acid,4-cyano-4-[(dodecylsulfanylthiocarbonyl)sulfanyl]pentanol,4-cyano-4-[(dodecylsulfanylthiocarbonyl)sulfanyl]pentanoic acid), andxanthates.

An ATRP initiator moiety includes any moiety that is capable ofinitiating a polymerization during ATRP process [17]. Examples of anATRP initiator moiety include, but not limited to, 2-bromopropanitrile(BPN), ethyl 2-bromoisobutyrate (BriB), ethyl 2-bromopropionate (EBrP),methyl 2-bromopropionate, 1-phenyl ethylbromide (1-PEBr), tosyl chloride(TsCl) , 1-cyano-1-methylethyldiethyldithiocarbamte (MANDC),2-(N,N-diethyldithiocarbamyl)-isobutyric acid ethyl ester (EMADC) , anddimethyl 2,6-dibromoheptanedioate (DMDBHD).

An NMP iniferter includes, but not limited to,2,2,6,6-tetramethylpiperidinyloxy (TEMPO) and TEMPO based derivatives.TEMPO based derivatives includes, but not limited to,4-acetamido-TEMPO-acetamido-2,2,6,6-tetramethylpiperidine 1-oxyl purum,4-amino-TEMPO, 2-azaadamantane-N-oxyl, 4-(2-bromoacetamido)-TEMPO,4-carboxy-TEMPO, 4-cyano-TEMPO, 4-hydroxy-TEMPO purum, 4-hydroxy-TEMPOpurum, 4-hydroxy-TEMPO benzoate, 4-(2-iodoacetamido)-TEMPO,4-isothiocyanato-TEMPO, 4-maleimido-TEMPO, 4-methoxy-TEMPO, 4-oxo-TEMPO,4-phosphonooxy-TEMPO hydrate,2,2,6,6-tetramethyl-4-(methylsulfonyloxy)-1-piperidinooxy.

A polymerization reaction of the present invention can be conducted byconventional methods such as mass polymerization, solutionpolymerization, suspension polymerization, emulsion polymerization,radiation polymerization (using γ-ray, electron beam or the like),radical polymerization, controlled-living radical polymerization (e.g.ATRP, RAFT polymerization, and NMP), photopolymerization, ring-openingpolymerization, and step-growth polymerization the like.

Polymerizing initiators include, but not limited to, electromechanicalradiation, thermal initiators, redox initiators and photoinitiators.Initiation of polymerization may be accomplished by irradiation withlight at a wavelength of between about 200 to about 700 nm. Redox-typeinitiators may be one or a combination of such initiators,tetramethylethylene, ferrous salt, sodium hydrogen sulfite or likereducing agent, etc. Examples of useful photoinitiators include2,2-dimethoxy-2-phenylacetophenone or a combination of ethyl eosin (10⁻⁴to 10⁻² M) and triethanol amine (0.001 to 0.1 M). Examples of thermalinitiators include, but not limited to, 4,4-azobis(4-cyanovaleric acid),benzoyl peroxide, azobisisobutyronitrile (AIBN), di-tertiary butylperoxide and the like. Such systems would initiate free radicalpolymerization at physiological temperatures include, for example,potassium persulfate, with or without tetraamethyl ethylenediamine;benzoylperoxide, with or without triethanolamine; and ammoniumpersulfate with sodium bisulfite.

The HA-based drug delivery platform is developed from encapsulation ofanesthetics, analgesics and antibiotics within a polymer matrix systemthrough physical interactions or chemical interaction (e.g. polymer-drugconjugation).

Physical interactions comprise any one of or a combination ofhydrophobic interaction, hydrophilic interaction, hydrogen bonding, andinter-molecular electrostatic interactions.

The polymer-therapeutic conjugation reactions comprise any one of or acombination of amine reaction, thiol reactions (e.g. thiol-ene clickreactions, Michael addition), carboxylate reaction, hydroxyl reactions,aldehyde and ketone reactions, active hydrogen reactions, photo-chemicalreaction, and cycloaddition reactions (e.g. diels-alder reaction, CuAAC,copper-free azide-alkyne huisgen cycloaddition).

The conjugated linkage comprise any one of or a combination ofisothiourea, isourea, amide, sulfonamide, shift-base, secondary amine,carbamate, arylamine, amidine, phosphoramidate, thioether, disulfide,β-thiosulfonyl, ester, carbamate, hydrazone, diazo, 2+4 cycloaddition,1,2,3-triazoles, carbohydrates, and amino acid esters bond.

EXAMPLES: Example 1

Materials

Hyaluronic acid sodium salt (HA) were purchased from Carbosynth Limited(Berkshire, UK). N-isopropylacrylamide (NIPAm) was purchased fromAldrich and was purified by recrystallization in hexane (3:1) beforeuse. S-1-dodecyl-S′-(α,α′-dimethyl-α″-acetic acid) trithiocarbonate(DATC) was synthesized according to the related reference [18].

Example 2

Synthesis of HA-g-PNIPAm Via the Combination of ReversibleAddition-Fragmentation Chain Transfer (RAFT) Polymerization andThiol-ene Click Reaction (FIG. 3 ).

The selection of a PNIPAm is based on its biocompatibility [19] andthermoresponsive phase transition characteristics that a hydrophiliccoils-hydrophobic globules transition occurs around 32° C. [20, 21]. Thecombination of RAFT polymerization to fabricate well-defined molecularweight polymers with the efficient coupling mechanism of thiol-ene“click” chemistry allows highly controlled formation of HA hydrogelswith distinct physical properties. The advantages of HA-g-PNIPAm areshown in FIG. 4 .

(1) Synthesis of HA-ADH.

HA (100 mg) was dissolved in 20 mL of water to prepare an HA solution of5 mg/mL. A 40 times molar excess of solid adipic acid dihydrazide (ADH)(1.736 g) was added to the solution and dissolved completely by mixingfor 10 min. The pH of the mixed solution was adjusted to 4.8 by theaddition of 1.0 N HCl. After that, four time molar excess of solid EDC(0.191 g) was added. The pH of the mixed solution was maintained at avalue of 4.8 by the addition of 1.0 N HCl. The reaction was stopped byraising the pH of the reaction solution to 7.0 with 1.0 N NaOH. Thereaction solution was dialyzed against a large excess amount of 100 mMNaCl solution, followed by dialysis against 25 vol % ethanol anddeionized water using a dialysis membrane (MWCO, 12-14 kDa). Theresulting solution was finally lyophilized for 3 days [13].

(2) Synthesis of HA-NAS.

100 mg HA-ADH was dissolved in 20 mL distilled water.N-acryloxysuccinimide (NAS) (0.5 g, 3 mmol) was subsequently added tothe HA-ADH solution. The reaction continued with stirring at roomtemperature for 12 h. HA-NAS was dialyzed extensively against 100 m MNaCl solution, followed by dialysis against 25 vol % ethanol anddeionized water using a dialysis membrane (MWCO, 12-14 kDa). The productwas then lyophilized for 3 days to obtain solid acrylated HA (HA-NAS).

(3) Synthesis of Carboxyl Terminated PNIPAm by RAFT Polymerization.

The mixture of NIPAm (3.0 g, 94 mmol), DATC (0.1000 g, 0.35 mmol), AIBN(10.0 mg, 0.0625 mmol) and DMF (5.0 mL) was placed in a 10 mLpolymerization tube. After oxygen was removed by purging argon, thesealed tube was immersed in a temperature controlled oil bath kept at60° C. and stirred for 24 h. After the heating was stopped, the reactionmixture was dissolved with THE and then precipitated in 10-fold diethylether. The polymer was collected by filtration and dried in a vacuumoven at 40° C. [21].

(4) Aminolysis of PNIPAm.

The THF solution of PNIPAm and hexylamine was reacted for overnight atroom temperature, the reaction mixture was precipitated from hexanes forthree times and resulting aminolysis product, thiolated PNIPAm(PNIPAm-SH).

(5) Conjugation of HA-NAS with PNIPAm-SH via a Thiol-ene Click Reaction.

The PNIPAm-SH and HA-NAS was then solubilized in deionized (DI) water.After stirring for overnight, the resulting solution was purified bydialysis (MWCO 50 kDa) against DI water. The product (HA-g-PNIPAm) wasthen recovered by freeze-drying as a white powder. The chemicalstructures of products in each steps are confirmed by fourier transforminfrared spectroscopy (FTIR) (FIGS. 6 ) and ¹H nuclear magneticresonance (NMR) spectroscopy (FIG. 7 ). The NMR spectra were obtained ona Bruker ARX 400 MHz. D₂O was used as a solvent for all the samples andthe reported spectra represented an average of 64 scans.

Example 3

Synthesis of HA-g-PNIPAm Via a ‘Grafting from’ Method Using RAFTPolymerization (FIG. 4 ).

(1) Synthesis HA-Based Macro RAFT Agent.

To render hyaluronic acid soluble in DMSO, the sodium ions of HA wereexchanged with the lipophilic tetrabutylammonium (TBA) ion. An aqueoussolution (1 L) of HANa (10 mg/mL) was subjected to ion-exchangecolumnchromatography (Dowex 50wx8[H+], Dow Chemicals, Midland, Mich.) toobtain an aqueous solution (1.5 L) of HA [22]. Next, tetrabutylammoniumbromide was added into 1000 mL of a 1% (w/w) HA solution in water andmixed for 2 h at room temperature. The mixture was then centrifuged for2 min at 5000 rpm to remove the resin. The obtained HA-TBA solution waslyophilized. The success of synthesis of HA-TBA was confirmed by FTIRand ¹H NMR. 1% (w/v) solution of HA-TBA in DMSO (100 mL) was prepared at50° C. under a nitrogen atmosphere. Subsequently, 0.2 g of DMAP and acalculated amount of DATC and DCC, depending on the requested degree ofsubstitution (DS), were added. The solution was stirred for 48 h at 50°C. HA-DATC was obtained by precipitation in diethyl ether for 3 times.The success of synthesis of HA-DATC was confirmed by UV-vis.

(2) RAFT Polymerization.

The polymerization conditions for the synthesis of HA-g-PNIPAmnanocomposite are as follows: HA-DATC: AIBN: PNIPAm=1:0.2:500 andreaction mixture. In detail, a Schlenk flask was added with 80 mg ofHA-DATC, 1 g of NIPAm, 2 mL of dry DMSO, and 0.55 mg of AIBN. Thereaction mixture was degassed by four freezes-pump-thaw cycles and thenwas placed in a shaker at 70° C. After the reaction time, thepolymerization was terminated by cooling via liquid nitrogen and thereaction mixtures were precipitated in 10-fold cold diethyl ether andwere dried at 40° C. in vacuum. The conversion was determinedgravimetrically.

Example 4

Hydrogel Formation and Gelation Time.

In brief, the HA-g-PNIPAm solutions (1-20 w/v %) at room temperaturewere quickly put in a water bath at 37° C. The time to form a gel(denoted as gelation time) is defined as the time when the gel, in aninverted state, shows no fluidity for 1 min [23]. The experiment wasperformed in triplicate. Table 1 indicates gelation temperature andgelation time of various concentration and molecular weight ofHA-g-PNIPAm.

TABLE 1 Gelation temperature and time of HA-g-PNIPAm hydrogels. SamplesGelation temperature (° C.) Gelation time (S) 15% HA-g- 32 30 PNIPAm 13k15% HA-g- 29 30 PNIPAm 5k 10% HA-g- 32 90 PNIPAm 13k

Example 5

Rheological Characterization.

Rheological experiments were carried out with a new Discovery SeriesHybrid Rheometer (DHR)-3 (TA) using parallel plate (20 mm diameter, 0°C.) configuration at 37° C. in the oscillatory mode. A 20 mm parallelplate geometry was used to perform strain sweeps and frequency sweeps at37° C. Time sweep were performed to determine the gelation time of thehydrogel. Each hydrogel sample was used for only one test. Each test wasperformed in triplicate and the data represents the average of the threetests with corresponding standard deviation. The testing time todetermine gelation time and modulus was very short and there wastherefore no need to use a humidified chamber or trap for theseexperiments.

HA-g-PNIPAm is soluble in water at room temperature but rapidly forms ahydrogel at physiological temperature over a range of its concentration(5-15% w/v) as evidenced by an inverse method (FIG. 8 and FIG. 9 ). Thehydrogel is able to retain its elasticity and shape when it iscompressed (FIG. 10 ). Rheology testing (FIG. 11 ) showed that increaseof the HA-g-PNIPAm concentration significantly increased the hydrogelstiffness. Accordingly, the elastic modulus of the 15% and 10%HA-g-PNIPAm hydrogels were ˜42 kPa and ˜32 kPa respectively indicatingtheir highly elastic properties presumably due to double hydrogenbonding network within the hydrogel (FIG. 12 ).

Example 6

In Vitro Drug Release

In vitro morphine release was evaluated in a membrane based diffusionsystem. HA-g-PNIPAm with different concentrations (5-15% w/v) andmorphine were prepared and loaded in a 3 mL syringe. The hydrogel wereequilibrated for 10 min in a 37° C. incubator. The hydrogel was thendispensed into cell culture inserts (12 mm diameter with 3μm pore size(Corning Incorporated, USA) in a 12 well plate. The hydrogels were thensubmerged with PBS and the well plate was placed into a 37° C. waterbath. At specified time intervals, 1 mL of the solution from individualwell was withdrawn and replaced with pre-heated water. Morphineconcentration from the buffer solution was determined by UVspectrophotometry at 263 nm.

A sustained morphine release profile was observed in 48 hours due todiffusion of morphine from morphine-loaded HA-g-PNIPAm hydrogels (FIG.13 ). Our hydrogel system also showed the feasibility to modulate therelease profile simply via tuning the concentration of HA-g-PNIPAmconcentration: after 48 hours, 15% HA-g-PNIPAM hydrogels had the slowestprofile with ˜70% cumulative release; 10% HA-g-PNIPAM hydrogelsdisplayed an intermediate release profile with ˜80% cumulative release.This result is consistent with the conclusion derived from the rheologydata. In another in vitro study, the hydrogels supported cellular growthof both chondrocytes and bone marrow-derived stem cells with highviability during culture, implying that our hydrogels arecytocompatible.

Example 7

In Vivo Drug Release through Subcutaneous Injections

In order to assess effectiveness of our hydrogel delivery system foranalgesia, a conscious rat model that allows for quantification ofmorphine release in plasma along with the evaluation of biocompatibilityof the hydrogels was employed. The specific protocols of in vivoefficacy assessment are designed as follows:

(1) Animal Population.

Rats (sample size n=10) were enrolled in the study. The study wasrandomized and blinded in pre-clinical trial. Each sprague dawley rat isassigned to 1 of 3 treatment groups including morphine-loaded hydrogels,positive control (morphine subcutaneous), negative controls(saline/hydrogel).

(2) Surgical Procedure.

Surgery under aseptic conditions was performed on individual rats.Isoflurane (3-5%) in anesthesia chamber was used for induction andmaintenance with isoflurane (1.5-3%) with mask. Dorsal and ventral neckwas shaved, scrubbed with Nolvasan, and swapped with alcohol. Both sideof the flanks were shaved at 1×1 square shape. In our pilot study,pockets of hydrogels were visible after 3 days confirming hydrogelformation on subcutaneous injections of macromolecule solution throughsyringe and possibly allowing for a sustained release over a period ofdays (FIG. 14 ).

(3) Pharmacokinetic Study.

Blood samples were withdrawn from subcutaneous port at designed timepoints using carotid artery placement method in a conscious rat modelafter 24 hours post carotid artery catheterization. The decanted plasmasubjected to SPE extraction. Additionally, a HPLC-mass spectrometry(HLPC-MS) method was identified for the reliable determination plasmalevel of morphine in rats. The analyte and internal standard(morphine-d3) were extracted from plasma samples by a single solid-phaseextraction (SPE) method prior to HPLC-MS. Our standard calibration graphis linear within a range of 10-1000 ng/mL (r=0.999). In vivo drugrelease of morphine from HA-g-PNIPAm hydrogels was shown in FIG. 15 . Invivo studies demonstrated HA-g-PNIPAm hydrogels enabled sustainedrelease of morphine above the therapeutic plasma concentration (long/mL)[24] up to 48 h (FIG. 15 ).

(4) Statistical Analysis.

Descriptive statistic and test of normality of data were done withShapiro Wilk test. Depending on the normality of the data, either ANOVAwill be employed (if data are normal) or Kruskal Wallis ANOVAstatistical test will be utilized (if nonparametric) to compare betweenthe treatment groups. Significance was set at P<0.05.

Example 8

In Vivo Drug Release Through Intra-Articular Injections

In vivo studies of the morphine-loaded novel hydrogels will be carriedout in canine animal models to assert the validity of this system inpain-relief studies and the duration of analgesic effect in dogs andpossibly man. Schematics of injectable hydrogels for intraarticulardelivery of an analgesic or anesthetic was shown in FIG. 16 . Thespecific protocols of in vivo efficacy assessment are designed asfollows [25]:

(1) Animals.

Client-owned dogs with cranial cruciate ligament rupture will beenrolled in the study with owner informed consent.

(2) Experiment Design.

Study design is prospective, randomized and blinded clinical trial. Arandomized number table will be used to assign dogs to 1 of 4 treatmentgroups for intra-articular administration of the following: salinesolution (control group), morphine group (control group), hydrogel only(control group), and morphine loaded hydrogel (treatment group).

(3) Procedure.

Prior to surgery all dogs will be premedicated with hydromorphoine (0.1mg/kg IM) and acepromazine (0.01 mg/kg IM) and anesthesia was inducedwith propofol (4-6 mg/kg, IV) and maintained with isoflurane (1-2.5% toeffect) in 100% oxygen. Routine tibial tuberosity advancement (TTA) ortibial plateau leveling osteotomy (TPLO) through a medial skin andarthrotomy approach will be conducted by a board-certified veterinarysurgeon or a surgical resident experienced in these techniques.

(4) Pain Assessment and Scoring.

All dogs will be assessed for signs of pain using modified criteriaadopted from two pain scoring system, the dynamic and interactive visualanalogue scale (DIVAS) for soft tissue surgery and the multifactorialpain score (MPS) developed for stifle arthrotomy study. The pain scoringassessment will be conducted at 2, 4, 6, 8, 10, 12, 16, 20, and 24 hoursfollowing the intra-articular injection by trained investigator, blindedto the intra-articular preparation used.

Rescue Parameter.

Dogs will be given rescue analgesia systemically if upon evaluation thescore system exceed: 70 mm FOR DIVAS; Glascow Scale composite pain scaleof 6.

Additional disclosure is found in Appendix-A, filed herewith, entiretyof which is incorporated herein by reference into the presentdisclosure.

Those skilled in the art will recognize that numerous modifications canbe made to the specific implementations described above. Theimplementations should not be limited to the particular limitationsdescribed. Other implementations may be possible. In addition, allreferences cited herein are indicative of the level of skill in the artand are hereby incorporated by reference in their entirety.

REFERENCES

[1] Uhrich K E, Cannizzaro S M, Langer R S, Shakesheff K M. PolymericSystems for Controlled Drug Release. Chemical Reviews. 1999; 99:3181-98.

[2] Hoare T R, Kohane D S. Hydrogels in drug delivery: Progress andchallenges. Polymer. 2008; 49:1993-2007.

[3] Bagshaw K R, Hanenbaum C L, Carbone E J, Lo K W, Laurencin C T,Walker J, et al. Pain management via local anesthetics and responsivehydrogels. Therapeutic delivery. 2015; 6:165-76.

[4] Calvert P. Hydrogels for Soft Machines. Advanced Materials. 2009;21:743-56.

[5] Julian T, Yujie M, Bruekers S M C, Shaohua M, Huck W T S. 25thAnniversary Article: Designer Hydrogels for Cell Cultures: A MaterialsSelection Guide. Advanced Materials. 2014; 26:125-48.

[6] Lee K Y, Mooney D J. Hydrogels for tissue engineering. Chem Rev.2001; 101:1869-79.

[7] Necas J, Bartosikova L, Brauner P, Kolar J. Hyaluronic acid(hyaluronan): a review. Veterinarni medicina. 2008; 53:397-411.

[8] Burdick J A, Prestwich G D. Hyaluronic Acid Hydrogels for BiomedicalApplications. Advanced Materials. 2011; 23 :H41-H56.

[9] Thiele J, Ma Y, Bruekers S M C, Ma S, Huck W T S. 25th AnniversaryArticle: Designer Hydrogels for Cell Cultures: A Materials SelectionGuide. Advanced Materials. 2014; 26:125-48.

[10] Bhatia D, Bejarano T, Novo M. Current interventions in themanagement of knee osteoarthritis. Journal of Pharmacy & BioalliedSciences. 2013; 5:30-8.

[11] Moskowitz R W. Osteoarthritis: diagnosis and medical/surgicalmanagement: Lippincott Williams & Wilkins; 2007.

[12] Mero A, Campisi M. Hyaluronic acid bioconjugates for the deliveryof bioactive molecules. Polymers. 2014; 6:346-69.

[13] Kirker K R, Luo Y, Nielson J H, Shelby J, Prestwich G D.Glycosaminoglycan hydrogel films as bio-interactive dressings for woundhealing. Biomaterials. 2002; 23:3661-71.

[14] Hu X, Li D, Zhou F, Gao C. Biological hydrogel synthesized fromhyaluronic acid, gelatin and chondroitin sulfate by click chemistry.Acta Biomaterialia. 2011; 7:1618-26.

[15] Grigoriadis C, Nese A, Matyjaszewski K, Pakula T, Butt H-J, FloudasG. Dynamic Homogeneity by Architectural Design—Bottlebrush Polymers.Macromolecular Chemistry and Physics. 2012; 213:1311-20.

[16] Motokucho S, Sudo A, Endo T. Polymer having a trithiocarbonatemoiety in the main chain: Application to reversibleaddition—fragmentation chain transfer controlled thermal andphotoinduced monomer insertion polymerizations. Journal of PolymerScience Part A: Polymer Chemistry. 2006; 44:6324-31.

[17] Matyjaszewski K. Atom Transfer Radical Polymerization (ATRP):Current Status and Future Perspectives. Macromolecules. 2012;45:4015-39.

[18] Lai J T, Filla D, Shea R. Functional polymers from novelcarboxyl-terminated trithiocarbonates as highly efficient RAFT agents.Macromolecules. 2002; 35:6754-6.

[19] Cooperstein M A, Canavan H E. Assessment of cytotoxicity of(N-isopropyl acrylamide) and Poly (N-isopropyl acrylamide)-coatedsurfaces. Biointerphases. 2013; 8:19.

[20] Eeckman F, Moes A J, Amighi K. Synthesis and characterization ofthermosensitive copolymers for oral controlled drug delivery. EuropeanPolymer Journal. 2004; 40:873-81.

[21] Hua D, Jiang J, Kuang L, Jiang J, Zheng W, Liang H. SmartChitosan-Based Stimuli-Responsive Nanocarriers for the ControlledDelivery of Hydrophobic Pharmaceuticals. Macromolecules. 2011;44:1298-302.

[22] Ohya S, Nakayama Y, Matsuda T. Thermoresponsive artificialextracellular matrix for tissue engineering: hyaluronic acidbioconjugated with poly (N-isopropylacrylamide) grafts.Biomacromolecules. 2001; 2:856-63.

[23] Cai S, Liu Y, Shu X Z, Prestwich G D. Injectable glycosaminoglycanhydrogels for controlled release of human basic fibroblast growthfactor. Biomaterials. 2005; 26:6054-67.

[24] Linares O A, Linares A L. Computational Opioid Prescribing: A NovelApplication of Clinical Pharmacokinetics. Journal of Pain & PalliativeCare Pharmacotherapy. 2011; 25:125-35.

[25] Soto N, Fauber A E, Ko J C, Moore G E, Lambrechts N E. Analgesiceffect of intra-articularly administered morphine, dexmedetomidine, or amorphine-dexmedetomidine combination immediately following stifle jointsurgery in dogs. Journal of the American Veterinary Medical Association.2014; 244:1291-7.

1. A polymer-based drug delivery platform comprising a compositioncomprising: a polymer matrix comprising: a functionalized hyaluronicacid (HA) of at least 100 monomeric units comprising a first functionalamino group with a theoretical substitution degree ranging from 20% to80% per monomeric unit of HA and a second functional group comprising apolymerizable moiety, and a telechelic polymer comprising a functionalgroup reactive to the second functional group of the HA, wherein: saidfunctionalized HA and said telechelic polymer are linked by a covalentbond formed between said second functional group of the HA and thefunctional group of the telechelic polymer, and gelation of said polymermatrix is reversible due to the presence of intra- and intermolecularhydrogen bonds; and the composition further encapsulates a protein, ananesthetic, an analgesic, or an antibiotic within the polymer matrixthrough physical interaction or chemical interaction.
 2. The platform ofclaim 1, wherein the physical interaction comprises any one of or acombination of hydrophobic interaction, hydrophilic interaction,hydrogen bonding, and inter-molecular electrostatic interactions.
 3. Theplatform of claim 1, further comprising an opioid or a nonsteroidalanti-inflammatory drug (NSAID).
 4. The platform of claim 1, wherein thepolymerizable moiety of the second functional group comprises anacrylate moiety.
 5. The platform of claim 1, wherein the theoreticaldegree of substitution of the first functional group results in at leastone unreacted amino group in the first functional group.
 6. The platformof claim 5, wherein the intra- and intermolecular hydrogen bonds areformed between isopropyl groups and the at least one unreacted aminogroups of the polymer matrix.
 7. The platform of claim 1, wherein thetelechelic polymer is thiolated poly (N-isopropylacrylamide) (PNIPAm).8. The platform according to claim 1, wherein the functionalized HAcomprises formula I, II, or IV:

wherein R₁, R₂, and R₅ each independently comprises any one of or acombination of haloacetates, hydrazides, amines, thiols, carboxylicacids, aldehydes, ketones, dienes, azide isothiocyanates, isocyanates,acyl azides, N-hydroxysuccinimide (NHS) esters, sulfo-NHS, sulfonylchloride, epoxides, carbonates, aryl halides, imidoesters,carbodiimides, alkylphosphate compounds, anhydrides, fluorophenylesters, hydroxymethyl phosphines, iodoacetyl derivatives, maleimides,aziridines, acryloyl derivatives, disulfide derivatives, vinylsulfone,phenylthioester, diazoacetates, carbonyl diimidazoles, oxiranes,N,N′-disuccinimidyl carbonates, N-hydroxylsuccinimidyl chloroformates,alkyl halogens, hydrazines, alkynes, and phosphorus-bound chlorine. 9.The platform of claim 1, wherein the polymerizable moiety of thefunctionalized HA comprises a methyl acrylate moiety.
 10. The platformof claim 7, wherein the telechelic polymer comprises a thiol group at_anend of each main chain of the PNIPAm and has a molecular weight rangingfrom 5 to 20 kDa.
 11. A method for controlled delivery of a drugcomprising: administering, to a subject, a polymer matrix comprising: afunctionalized hyaluronic acid (HA) of at least 100 monomeric unitscomprising a first functional amino group with a theoreticalsubstitution degree ranging from 20% to 80% per monomeric unit of HA anda second functional group comprising a polymerizable moiety, and atelechelic polymer comprising a functional end-group reactive to thesecond functional group of the HA, wherein: said functionalized HA andsaid telechelic polymer are linked by a covalent bond formed betweensaid second functional group of the HA and the functional end-group ofthe telechelic polymer, and gelation of said polymer matrix isreversible due to the presence of intra- and intermolecular hydrogenbonds; and wherein the polymer matrix further encapsulates a drugthrough physical interaction or chemical interaction; wherein the drugcomprises a protein, an anesthetic, an analgesic, or an antibiotic. 12.The method of claim 11, further comprising the steps of: incorporatingthe drug in the polymer matrix at a drug concentration ranging from 0.1%to 5% (w/v); and inducing the gelation for subsequent drug release byexposing polymer matrix-drug aqueous precursor solution to a temperaturehigher than a gelation temperature of the polymer matrix aqueousprecursor solution or a physiological temperature of a human.
 13. Themethod of claim 12, further comprising the step of preparing the polymermatrix aqueous precursor solution below gelation temperature, wherein aconcentration of polymer matrix in the aqueous precursor solution rangesfrom about 5 to about 15% (w/v).
 14. The method of claim 12 furthercomprising adjusting a concentration of polymer matrix in the aqueousprecursor solution and/or a molecular weight of the polymer matrix tomodify a rate at which the polymer matrix releases the drug, wherein thedrug release rate decreases with an increase in the concentration ofpolymer matrix in the aqueous precursor solution and/or an increase inthe molecular weight of the polymer matrix.
 15. The method of claim 11,wherein the polymer matrix comprises HA-g-PNIPAm, the drug is morphine,and a concentration of HA-g-PNIPAM in the aqueous precursor solutionranges from 5-15% (w/v).
 16. The method of claim 15, wherein themorphine concentration in the aqueous precursor solution is 0.468%(w/v).
 17. The method of claim 11, wherein the physical interactioncomprises any one of or a combination of hydrophobic interaction,hydrophilic interaction, hydrogen bonding, and inter-molecularelectrostatic interactions.
 18. The method of claim 11, wherein the drugcomprises an opioid or a nonsteroidal anti-inflammatory drug (NSAID).19. The method of claim 11, wherein: the polymerizable moiety of thesecond functional group comprises an acrylate moiety; and thetheoretical degree of substitution of the first functional group resultsin at least one unreacted amino group in the first functional group;wherein the intra- and intermolecular hydrogen bonds are formed betweenisopropyl groups and the at least one unreacted amino groups of thepolymer matrix.
 20. The method of claim 11, wherein administeringcomprises implantation of the polymer matrix, or intra-articular orsubcutaneous injection of the polymer matrix.
 21. The method of claim11, wherein the drug comprises a protein in the form of bovine serumalbumin.